Eclipse ti u inverted research microscope

The Eclipse Ti-U inverted research microscope (Nikon, Tokyo, Japan) with a Nikon Digital Sight DS-Fi1 camera system was used to take brightfield microscopy photographs of cells of each Dunaliella strain. The objective lens used was Nikon Splan Fluor ELWD 60×/0.7 and the ocular lens was Nikon CFI 10×/22. The NIS-Elements Advanced Research Microscope Imaging Software (Nikon, Tokyo, Japan) was used to acquire the photos. Differential interference contrast (DIC) microscopy photographs were also obtained using a confocal microscope system ZEISS LSM 880 (Carl Zeiss Microscopy GmbH, Jena, Germany). The ZEISS Plan Apochromat 63×/1.4 oil DIC objective lens and the Carl Zeiss PI 10×/23 ocular lens were used. Images were acquired and analyzed through the ZEN 2.1 LSM software (Carl Zeiss Microscopy GmbH).

EndoMT was induced by changing medium to an EndoMT-inducing medium that contained Stempro34 medium with Stempro34 supplement (Life Science Technology Corp.), L-glutamine (Life Technologies Corp.), penicillin–streptomycin (Life Technologies Corp.), 200 ng ml⁻¹ BMP2 (Peprotech Inc.) and 50 ng ml⁻¹ TGFβ2 (Peprotech Inc.). Non-EndoMT control groups were kept in EC medium containing Stempro34 (Life Technologies Corp.), Stempro34 supplement (Life Technologies Corp.), L-glutamine (Life Technologies Corp.), penicillin–streptomycin (Life Technologies Corp.) and 50 ng ml⁻¹ vascular endothelial growth factor (Peprotech Inc.). Cells were collected 3 days after induction.
Type I collagen (Sigma Corp.) at 1 mg ml⁻¹ (final concentration) was mixed with Stempro34 medium, Stempro34 supplement (Life Science Technology Corp.) and 50 mM NaOH (Sigma Corp.). The mixture was poured into 24-well tissue culture plates (0.5 ml per well) and allowed to gel in 5% CO₂ incubator at 37 °C for 30 min. And then 0.5 ml EndoMT-inducing medium was added. After 3 days, pictures of cells were taken with 100 times magnificance under the Eclipse Ti-U inverted research microscope (Nikon Corp.). Mesenchymal cells that migrated out in three pictures from different fields were counted. Experiments were repeated three times. Average value and standard derivation were plotted.